PROJECT SUMMARY Mutations in KRAS are among the most frequent in cancer, particularly in certain poor prognosis subtypes such as lung and pancreatic adenocarcinomas. Extensive evidence suggests that KRAS is a cancer driver and most tumors remain addicted to this oncoprotein. Biochemically, KRAS acts as a molecular binary switch that cycles between a GTP-bound active state and a GDP-bound inactive state and KRAS oncogenic mutations favor the active conformation. In the active state, KRAS associates with protein effectors that propagate the downstream signal and contribute to the malignant phenotype. In this proposal, I describe the discovery of Sin1 as a novel potential KRAS effector and hypothesize its role in contributing to the initiation of KRAS-driven malignancies. Through this application, I plan a series of advanced biochemical and structural experiments aimed at determining whether Sin1 acts as a canonical Ras effector. Given the essential role of Ras effectors during tumorigenesis, this project will also study the effect of Sin1 depletion in animal models and organoid cultures transformed by an activated form of KRAS. Previous studies have shown that Sin1 is an essential component of the mTOR complex 2 (mTORC2), which regulates kinase phosphorylation, metabolism, and is itself central to proliferative signaling in cancer, but the contribution of KRAS in this context has not been established at the molecular level. Moreover, early studies in model organisms have shown that Sin1 is a component of the stress mitogenic pathways by interacting with MEKK2 and JNK. This proposal will address how Sin1 may act at the intersection of these two pathways and KRAS: I will determine, through the use of specific Sin1 separation-of-function mutants, whether the mechanisms of this potential KRAS effector are mTORC2-dependent or independent. If successful, the results of this project will have a significant impact in the field of KRAS signaling in particular and the cancer biology in general: the characterization of a novel KRAS-specific effector critical for tumorigenesis. The results of this study could provide a strong rationale for novel therapeutic approaches targeting previously uncharacterized KRAS effectors, a potential boon for patients with KRAS-driven malignancies. As part of my career development, I have put together a specific plan that includes activities for my mentored phase (K99) that are aimed at facilitating my transition to an independent principal investigator position (R00), including: a) Discussion with supervisor and advisory panel; b) Experience from personally conducting the research; c) Mentoring experience; d) Institutional developmental resources and coursework; and e) Online learning resources and bibliography. These activities will also be complemented by a strong mentorship from the cancer researchers Dr. Frank McCormick, who will advise on the biochemistry, and the members of my advisory panel Dr. Allan Balmain, who will advise on KRAS mouse models, and Dr. Calvin Kuo, who will advise on the organoid work. They have committed to provide technical, conceptual, and mentoring support. Two additional activities will be carried out during my K99 phase to complement my research skills; the first will involve learning how to isolate, culture, and work with organoids derived from different organs; while the second will be a conceptual and technical training in mass spectrometry in collaboration with Dr. Anatoly Urisman, who will help identify key interactors and effectors of Sin1. This award would provide me with the necessary tools, time, and resources to gain deeper insights into the biology of KRAS and Sin1 signaling, as well as fostering the necessary collaborations and skills to transition into an independent academic career.